Stem cell therapies for various degenerative diseases and traumatic injuries may be capable of restoring cell loss and stimulating tissue regeneration. Pluripotent embryonic stem cells (ESCs) are capable of differentiating into functional neurons, cardiomyocytes and pancreatic beta cells, thus representing a robust cell source for the development of an array of different regenerative cellular therapies. Despite the clear potential of pluripotent stem cells, a critical limitation is the inability to spatially and temporally control the molecular delivery of morphogenic factors to ESCs in a manner similar to embryonic development that efficiently directs the differentiation of the cells to specific phenotypes. Microspheres made of different biomaterials can be engineered to locally control the release of bioactive molecules, thereby simultaneously providing spatial, temporal and dose-dependent control of biomolecular delivery to cells and tissues. The integration of engineered microspheres into stem cell microenvironments for controlled, local delivery of morphogens to stem cells during the course of differentiation is a novel and translatable approach for the development of directed stem cell regenerative therapies. The objectives of this proposal are to 1) examine the effects of incorporating different types of microspheres within aggregates of ESCs undergoing differentiation referred to as embryoid bodies (EBs), 2) determine the spatiotemporal effects of independently delivering morphogens, such as Wnt 3a and BMP2, within EB microenvironments, and 3) examine the effects of sequential delivery of different morphogens (Wnt3a and BMP2) from microspheres incorporated within EBs. The completion of these studies will yield novel information about the ability to spatially and temporally control the molecular composition of the extracellular microenvironment regulating ESC differentiation via biomaterials-based delivery methods. In addition, this approach represents a fundamentally new route to directing the differentiation of stem cells in vitro through the controlled presentation of molecular factors locally, which may be a broadly applicable principle in the development of stem cell technologies. PUBLIC HEALTH RELEVANCE: Stem cell-based therapies seeking to restore viable cells to diseased or wounded tissues could promote functional tissue regeneration, but are currently limited by an inability to efficiently control the differentiation of the stem cells. This proposal seeks to improve the efficiency and homogeneity of pluripotent stem cell differentiation by spatially and temporally controlling the presentation of morphogenic factors regulating stem cell differentiation from microspheres incorporated within 3D stem cell microenvironments.